We have used the purine biosynthetic pathway as a paradigm for thinking about the importance of transient protein/protein interactions in the direct transfer (channeling) of chemically unstable intermediates between successive enzymes in metabolic pathways. The over-expression of all the enzymes in this pathway from E. coli and the availability of structures of 6 of the 11 enzymes from E. coli, make this an excellent pathway to examine the channeling question, mechanistic questions, as well as questions about the evolution of a pathway. The product of PurF, the first enzyme in the pathway, is the chemically unstable phosphoribosylamine (PRA). Our in vitro kinetic studies have suggested that PRA is channeled directly to PurD, the second enzyme in the pathway. The availability of structures of PurF from E. coli and B. subtilis and of PurD from E. coli have allowed us to propose a structural model for the channeling of PRA uniquely between enzymes from the same organism. In addition to the availability of the E. coli PurF and PurD, we now have available the B. subtilis PurF and PurD. Kinetic studies in vitro between these proteins from heterologous sources as well as between the two B. subtilis proteins will be examined. Our model predicts that only the ternary complexes of PurF.PRA.PurD from the same organism should channel. The available structures allow us to attach site specifically fluorescent probes to look for the transient interactions between these proteins in vitro and ultimately in vivo. In addition, using homologous recombination methods, the B. subtilis purF ORF will be manipulated to precisely replace the E. coli purF ORF in wild type E. coli. The phenotypic consequences of this replacement will be examined. We have recently obtained the structures of PurK and PurE, the sixth and seventh enzymes in this pathway. We have proposed, that these enzymes as well might channel the chemically unstable N5-CAIR, the product of PurK. Similar experiments to those described for PurF and PurD can now be carried out with PurK and PurE. Finally in addition to PurD, PurK and PurE, we have also obtained the structure of PurM, the 5th enzymes in this pathway. At present most of the structures are wild-typ4e, that is nucleotides are bound. To increase the insight that these structures will provide about function, we will synthesize a variety of potential inhibitors of these enzymes. The liganded structures should provide more specific insight about mechanistic possibilities. The structures have already suggested a variety of mechanistic experiments that will be carried out. The commonality of the ribose-5 P moiety of all the intermediates in this pathway, the fact that PurD, PurK and PurT are structurally homologous and catalyze similar chemistries, and that PurL and PurM appear also to be structurally and mechanistically related, provides food for thought about the evolution of a biosynthetic pathway.